The present invention relates to biological compounds, and genes encoding biological compounds, for use as pesticides, as well as methods for obtaining such compounds.
Unfortunately, it has increasingly been seen over the past several decades that employment of conventional chemical insecticides often leads to undesirable environmental consequences. Such consequences include toxicity to non-target organisms such as birds and fish, and human health hazards. Furthermore, pesticide management in the United States and elsewhere in the world is becoming increasingly complicated due to the evolution of insect resistance to classical chemical pesticides. Despite over 10 billion dollars being spent each year to control phytophagus insects, global losses in the food supply due to insects is still estimated to be about 20 to 30 percent (See, Oerke, Estimated crop losses due to pathogens, animal pests and weeds, 72-78 in Crop production and crop protection: Estimated losses in major food and cash crops (Elsevier, Amsterdam 1994)). There remains, therefore, an urgent need to develop or obtain substances that can be used safely in the fight against insect pests.
Over the past several years, there have been proposed a number of xe2x80x9cenvironmentally friendlyxe2x80x9d strategies to combat highly resistant insect pests such as certain species of cotton bollworm (e.g., Helicoverpa zea).
One recently introduced approach to insect management is the production of transgenic crops that express insecticidal toxins, such as engineered potato and cotton crops that express protein toxins from the soil bacterium Bacillus thuringiensis (Estruch, J. J. et al., Transgenic plants: An emerging approach to pest control, Nature Biotechnology 15, 137-141, 1997).
A variation of this strategy is the release of insect-specific viruses that have been genetically engineered to express insecticidal neurotoxins (Cory, J. S. et al., Field trial of a genetically improved baculovirus insecticide, Nature 370, 138-140, 1994). Baculoviruses, for example, are arthropod-specific viruses with no member of the baculovirus family known to infect either vertebrates or plants. The infectivity of some baculoviruses is restricted to a few closely related species within a single family of lepidopterous insects (moths and butterflies) (See, e.g., U.S. Pat. No. 5,639,454). Some baculoviruses, such as the beet armyworm nuclear polyhedrosis virus, target only a single species. As a result of their high degree of specificity, baculoviruses have long been envisaged as potential pest control agents and were first used as such in the 1970s. Their specificity means that baculoviral insecticides complement natural predators, rather than replacing them, as is the case with many chemical insecticides. However, to date, baculoviruses have met with only limited commercial success. Most naturally occurring baculoviruses take 4-7 days to kill their hosts, with some species taking considerably longer. During this time the insect continues to feed and cause crop damage, thus limiting the ability of baculoviral insecticides to compete with chemical agents.
This shortcoming has been addressed by engineering recombinant baculoviruses that express insect-specific neurotoxins. Expression of heterologous insect toxins not only reduces the time interval between virus application and insect death, but also reduces the mean feeding time (Prikhod""ko et al., Effects of simultaneous expression of two sodium channel toxin genes on the properties of baculoviruses as biopesticides, Biological Control 12, 66-78, 1998). Importantly, introduction of genes for insect-selective toxins does not alter the intrinsic infectivity of the baculovirus or its natural host range (Black et al., Commercialization of baculoviral insecticides, in The Baculoviruses (ed. Miller, L. K.) 341-387 (Plenum Press, New York, USA, 1997)).
New approaches to insect-pest management have stimulated interest in peptide toxins from the venoms of animals, particularly spiders and scorpions, that prey on insect species.
Zlotkin et al., An Excitatory and a Depressant Insect Toxin from Scorpion Venom both Affect Sodium Conductance and Possess a Common Binding Site, Arch. Biochem and Biophysics 240, 877-887, 1985), disclose two insect selective toxins from the venom of the scorpion Leiurus quinqestriatus, one of which induced fast excitatory contractive paralysis of fly larvae while the other induced slow depressant flaccid paralysis, with both affecting sodium conductance in the neurons. Likewise, Canadian patent 2,005,658 (issued: Jun. 19, 1990 to Zlotkin et al.) discloses an insecticidally effective protein referred to as xe2x80x9cLqhP35xe2x80x9d derived from the scorpion Leiurus quinquestriatus hebraeus. 
A number of investigators have also recognized spider venoms as a possible source of insect-specific toxins for agricultural applications (See, Jackson et al., Ann. Rev. Neurosci. 12, 405-414 (1989)). For example, U.S. Pat. Nos. 4,855,405 (issued: Aug. 8, 1989 to Yoshioka et al.) and 4,918,107 (issued: Apr. 17, 1990 to Nakajima et al.) both disclose glutamate-receptor inhibitors obtained from the venom of spiders as possible insecticidal agents. In U.S. Pat. Nos. 5,457,178 (issued: Oct. 10, 1995), 5,695,959 (issued: Dec. 9, 1997), and 5,756,459 (issued: May 26, 1998), Jackson et al. disclose a family of insecticidally effective proteins isolated from the venom of the spiders Filistata hibernalis (a common house spider) and Phidippus audax (a xe2x80x9cjumping spiderxe2x80x9d).
A particular group of spiders which has generated considerable investigative interest are the finnel-web spiders. WO 89/07608 (published: Aug. 24, 1989, Cherksey et al.) discloses low molecular weight factors isolated from American funnel-web spider venoms which reversibly bind to calcium channels. Adams et al., Isolation and Biological Activity of Synaptic Toxins from the Venom of the Funnel Web Spider, Agelenopsis aperta, in Insect Neurochemistry and Neurophysiology, Borkovec and Gelman (eds.) (Humana Press, New Jersey, 1986) teaches that multiple peptide toxins which antagonize synaptic transmission in insects have been isolated from the spider Agelenopsis aperta. In WO 93/15108, a class of peptide toxins known as the xcfx89-atracotoxins are disclosed as being isolated from the Australian funnel-web spiders (Araneae:Hexathelidae:Atracinae) by screening the venom for anti-Helicoverpa (xe2x80x9canti-cotton bollwormxe2x80x9d) activity. Such toxins are disclosed to have a molecular weight of approximately 4000 amu, to be of 36-37 amino acids in length, and capable of forming three intrachain disulfide bridges. One of these compounds, designated xcfx89-ACTX-Hv1 has been shown to selectively inhibit insect, as opposed to mammalian, voltage-gated calcium channel currents (Fletcher et al., The structure of a novel insecticidal neurotoxin, xcfx89-atracotoxin-Hv1, from the venom of an Australian funnel web spider, Nature Struct. Biol. 4, 559-566 (1997)). Homologues of xcfx89-ACTX-Hv1 have been isolated from the Blue Mountain funnel-web spider Hadronyche versuta (See, Wang et al., Structure-function of xcfx89-atrocotoxin, a potent antagonist of insect voltage-gated calcium channels, Eur. J. Biochem. 264, 488-494 (1999)).
While some insecticidal peptide toxins isolated so far from scorpions and spiders offer promise, there still remains a significant need for compounds which display a wide differential in toxicity between insects and non-insects, and yet which have significant insecticidal activity and a quick action.
The present inventors have isolated, and structurally and functionally characterized, a novel insecticidal toxin, designated xcfx89-atracotoxin-Hv2a, from the venom of the Australian funnel-web spider H. versuta. This toxin is a highly potent and specific antagonist of insect calcium channels. The toxin of the present invention shows no significant sequence similarity to any previously isolated insecticidal toxins, and it shows no sequence or structural homology with the omega-atracotoxin-Hv1 family of insecticidal toxins previously isolated from H. versuta (See, Atkinson et al., Insecticidal toxins derived from funnel web spider (Atrax or Hadronyche) spiders, PCT/AU93/00039 (WO 93/15108) (1993); Fletcher et al., The structure of a novel insecticidal neurotoxin, xcfx89-atracotoxin-HV1, from the venom of an Australian funnel web spider, Nature Struct. Biol. 4, 559-566 (1997); Wang et al., Structure-function studies of xcfx89-atracotoxin, a potent antagonist of insect voltage-gated calcium channels, Eur. J. Biochem. 264, 488-494 (1999)).
The present invention teaches the use of xcfx89-atracotoxin-Hv2a, or the gene coding for the toxin, as a biopesticide, either alone or in combination with other insecticidal toxins or genes thereof. It further teaches the use of the toxin, or the gene coding for the toxin, as a screen for natural or non-natural compounds that specifically inhibit insect calcium channels. Furthermore, the present invention provides in the determination of the toxin""s three-dimensional structure, a model for developing non-peptidic mimics of the toxin that could be used as foliar pesticide sprays.
In a first embodiment of the present invention, there is provided a polypeptide toxin that is toxic to adult and/or larval insects having a molecular mass of approximately 4,478 Daltons and a length of 45 amino acid residues. The polypeptide is capable of forming three intrachain disulfide bonds. Activity may be demonstrated by rapid paralysis of insects and/or potent inhibition of whole-cell calcium currents in isolated insect neurons. Phylogenetic specificity may typically be demonstrated by minimal activity in rat or chicken nerve-muscle preparations and/or minimal antagonism of calcium channel currents in isolated rat neurons.
The preferred toxin of the present invention is omega-atracotoxin-Hv2a (SEQ ID NO:1), abbreviated as omega-ACTX-Hv2a or xcfx89-ACTX-Hv2a, as defined herein:
SEQ ID NO:1: Leu-Leu-Ala-Cys-Leu-Phe-Gly-Asn-Gly-Arg-Cys-Ser-Ser-Asn-Arg-Asp-Cys-Cys-Glu-Leu-Thr-Pro-Val-Cys-Lys-Arg-Gly-Ser-Cys-Val-Ser-Ser-Gly-Pro-Gly-Leu-Val-Gly-Gly-Ile-Leu-Gly-Gly-Ile-Leu
The toxins of the present invention may be isolated from spider venom or chemically synthesized and oxidized/folded using similar techniques to those described previously for production of synthetic omega-atracotoxin-Hv1a (See, Atkinson et al., Insecticidal toxins derived from funnel web spider (Atrax or Hadronyche) spiders, PCT/AU93/00039 (WO 93/15108) (1993); Fletcher et al., The structure of a novel insecticidal neurotoxin, xcfx89-atracotoxin-HV1, from the venom of an Australian funnel web spider, Nature Struct. Biol. 4, 559-566 (1997), both of which are incorporated by reference in their entirety herein). The toxin could also be prepared from a synthetically constructed gene using recombinant DNA techniques as the authors have described previously for an unrelated protein (Riley et al., Cloning, expression, and spectroscopic studies of the Jun leucine zipper domain, Eur. J. Biochem. 219, 877-886 (1994) which is incorporated in its entirety herein). A DNA probe coding for the amino sequence of the toxin may be used to isolate the gene coding for the protein or the corresponding preprotein or preproprotein using standard molecular biological techniques. The natural or synthetic gene(s) may be inserted into appropriate overexpression vectors for production of the toxin. In particular, the gene for the protein, preprotein, or preproprotein may be inserted into the genome of an appropriate insect vector, such as a baculovirus. Alternatively, transgenic plants may be constructed that express the toxin or the preprotein or preproprotein form of the toxin. Thus, the invention also provides insect viruses and plant species engineered to express the toxins of this invention.
In another embodiment of the present invention, there is provided variants of xcfx89-ACTX-Hv2a, wherein a xe2x80x9cvariantxe2x80x9d is defined as a polypeptide that corresponds to or comprises a portion of xcfx89-ACTX-Hv2a, or is homologous to xcfx89-ACTX-Hv2a. For the purposes of this invention, xe2x80x9chomologyxe2x80x9d between two peptide sequences connotes a likeness short of identity, indicative of a derivation of the first sequence from the second. In particular, a polypeptide is xe2x80x9chomologousxe2x80x9d to xcfx89-ACTX-Hv2a if a comparison of their amino acid sequences reveals an identity greater than about 30% (which is usually sufficient to indicate structural homology). Such a sequence comparison can be performed via numerous computer algorithms in the public domain.
In yet another embodiment of the present invention, there is provided a method of screening for, or designing, an antagonist of insect calcium channels. This method involves selecting or designing a substance which inhibits the binding of xcfx89-ACTX-Hv2a, or a variant thereof, to insect calcium channels and testing the ability of the substance to act as an antagonist of insect calcium channels. The term xe2x80x9cinsect calcium channelxe2x80x9d refers to any insect calcium channel that is inhibited by xcfx89-ACTX-Hv2a.
There is also provided by the present invention, a method of screening for substances for insecticidal potency and phylogenetic specificity, the method comprising: (a) measuring the ability of a substance to inhibit the binding of xcfx89-ACTX-Hv2a, or a variant thereof, to insect calcium channels; (b) measuring the ability of the substance to antagonize insect calcium channels; and (c) determining whether the substance has minimal activity against vertebrate calcium channels. Preferably the substance isolated by use of such method has high phylogenetic specificity being defined herein as greater than 100-fold selectivity for insect over vertebrate calcium channels, and preferably greater than 1000-fold selectivity for insect over vertebrate calcium channels.
According to yet another embodiment of the present invention there is provided an insecticidal composition for delivering xcfx89-ACTX-Hv2a, a variant thereof, or an inhibitor of insect calcium channels, discerned by the methods described above. For example, where the toxin, variant, or calcium channel antagonist can be expressed by an insect virus, the virus encoding the toxin, variant, or calcium channel antagonist can be applied to the crop to be protected. The virus may be engineered to express xcfx89-ACTX-Hv2a, a xcfx89-ACTX-Hv2a variant, or one of the calcium channel inhibitors either alone, in combination with one another, or in combination with other insecticidal polypeptide toxins that may result in synergistic insecticidal activity. The virus may be formulated in an agriculturally acceptable carrier, diluent and/or excipient. Suitable viruses include, but are not limited to, baculoviruses.
Alternatively, the crop itself may be engineered to express xcfx89-ACTX-Hv2a, a xcfx89-ACTX-Hv2a variant, or a calcium channel antagonist, discerned by the above described methods, either alone, in combination, or in combination with other insecticidal polypeptide toxins that may result in synergistic insecticidal activity. Crops for which this approach would be useful include cotton, tomato, green bean, sweet corn, lucerne, soybean, sorghum, field pea, linseed, safflower, rapeseed, sunflower, and field lupins.
Alternatively, the insecticidal agent may be delivered directly to the crop in an agriculturally acceptable carrier, diluent and/or excipient. Delivery could, for example, be in the form of a foliar spray. Insect infestation of crops may be controlled by treating the crops and/or insects with such compositions. The insects and/or their larvae may be treated with the composition, for example, by attracting the insects to the composition with an attractant.